The activity of cytokine is associated with pathologic severity of inflammatory and for immune response to various antigenic stimulations. Many antigen specific antibodies and soluble receptors which could recognize cytokines are currently in use to inhibit the function of cytokines for the therapeutic purposes (WO 93/016184, WO 96/02576, WO 96/023067, WO 1997/03682, and U.S. Pat. Nos. 5,434,131, 5,656,272, 5,977,318, 6,210,661, 6,225,117). Antibodies and soluble receptors inhibit cytokine signal transduction by disturbing interaction between cytokines and their receptors on cell surface.
Soluble receptors used as functional inhibitors of cytokine that fused to heavy chains of human immunoglobulins were disclosed by Capon et al. (Nature 337:5254, 1989), and thereafter many patents were disclosed inventions related to fusion proteins of soluble receptors and immunoglobulins (U.S. Pat. Nos. 5,521,288, 5,844,095, 6,046,310, 6,090,914, 6,100,383, 6,225,448).
Generally, fusion proteins of soluble receptors and immunoglobulins have following advantages (Capon et al., Nature 337:5254, 1989)
1. Increase in total avidity to ligand by forming bivalency via dimerization.
2. Increase in blood half-life of proteins, that is, increase in molecular stability
3. Activation of effecter cells by Fc fragment of immunoglobulin heavy chain
4. Convenience of purification by using affinity column, e.g. using protein A
Most fusion proteins of receptor extracellular domain and immunoglobulin heavy chain are composed of heavy chain without CH1 domain, which result in dimers not binding to light chains. This structure is more desirable for the function of proteins and receptors involving immune response. For example, TNFR(WO92/16221, WO95/34326)-immunoglobulin fusion proteins disclosed in WO94/06476 and U.S. Pat. No. 5,447,851 have been used for the inhibition of TNF-mediated inflammation. It is well known that TNFR-immunoglobulin fusion proteins have a higher affinity than original monomeric molecules (Lesslauer et al., Eur. J. Immunol. 21:2883, 1-991; Ashkenazi et al., Proc. Natl. Acad. Sci. 88:10535, 1991; Peppe et al, J. Exp. Med. 174:1483, 1991; Mohler et al., J. Immunol. 151:1548, 1993).
For the improved inhibition of TNF mediated response, one can increase efficacy by multimerizing soluble extracellular domains of TNFR, CD2, and CTLA-4. For example, when fusion proteins of TNFR's extracellular domains bound with immunoglobulin heavy chain (heavy chain fusion protein) and with light chain (light chain fusion protein) respectively are coexpressed in the same cell, one can produce fusion proteins as a tetrameric form by linking heavy chain to heavy and light chains. This tetramer showed much more increased efficacy than monomeric or dimeric forms as presented by Scallon et al. (Cytokine 7:759, 1995).
However, this method had many difficulties for commercialization such as simultaneous expression of two different fusion genes in the same cell line, remarkably lower production yields of multimeric form; and difficulty in purifying multimeric high molecular weight forms. For these reasons, immunoglobulin fusion proteins currently in use are only heavy chain fused form.
Therefore, there is considerable demand for the development of methods of producing multimeric protein therapeutics with high yield and efficient purification procedures.